Testosterone and estradiol predict male calling performance, but not performance-related tradeoffs, in competitive signaling environments in Cope’s gray treefrogs (Hyla chrysoscelis)

Acoustic advertisement signals in anurans are classic models for understanding how endocrine mechanisms regulate courtship behavior. However, our understanding of how steroids influence male calling performance remains limited because most studies examine single hormones or isolated call traits. Here we quantified three plasma steroids—testosterone (T), estradiol (E2), and corticosterone (CORT)—from male Cope's gray treefrogs ( Hyla chrysoscelis ) recorded in natural male–male calling pairs, relating these concentrations to multiple calling components including call rate, duration, and effort. We used principal components analysis to describe a male's overall calling performance and his calling strategy (how he navigated a performance tradeoff between longer calls and faster rates). We analyzed these relationships at the population level and within male pairs using multiple regressions including all three hormones as predictors. At the population level, T and E ₂ positively correlated with calling performance, whereas calling strategy was unrelated to hormones. Within male pairs, differences in T also positively correlated with differences in calling performance. Despite their collinearity, T and E ₂ independently and together explained variation in calling performance. CORT showed no association with calling performance or strategy at either level, and temperature and body condition did not explain variation in calling or hormones. These results show that gonadal activation translates into asymmetries in calling performance between competitors while leaving performance-related tradeoffs unaffected. By revealing strong positive correlations between gonadal hormones and energetic investment in calling, this study demonstrates how female preferences for high-performing callers can impose sexual selection on endocrine mechanisms regulating courtship behavior.